anti-PA Antibody Research Articles

Binding specificity and affinity analysis of an anti-protective antigen peptide reagent using capillary electrophoresis

Peptide biosensor reagents are emerging as an alternative to typical antibody-based detection methods. Peptides can be rapidly isolated using bacterial display methods for new and emerging biothreats and can be chemically synthesized for rapid, large-scale production. With the emergence of peptide biosensor reagents, there is a growing need to develop methods for characterizing binding interactions. Capillary electrophoresis (CE) is a free-solution separation method that is able to determine target and analyte binding association (Kb) and dissociation constants (Kd). In this study, the Kb, Kd, and peptide specificity of an isolated peptide binding reagent to protective antigen (PA) of Bacillus anthracis were evaluated using capillary electrophoresis at 10 and 20 kV. The relative binding specificity was rapidly assessed by measuring the peptide relative mobility shift at 20 kV at nonequilibrium using bovine serum albumin (BSA), horseradish peroxidase (HRP), and an anti-PA monoclonal antibody (mAb). The αPA peptide was shown to be highly specific for PA, with a Kd = 177 nM measured at 20 kV and Kd = 312 nM measured at 10 kV. These results show that peptides from bacterial display libraries can be rapidly tested for specificity and binding affinity, in solution, for use as a potential biosensor reagent against new and emerging biothreats.

In vitro and in vivo activities of recombinant anthrax protective antigen co-expressed with thioredoxin in Escherichia coli

Because of the central role it plays in the formation of lethal toxin and edema toxin, protective antigen (PA) is the principal target for the development of vaccines against anthrax. In the present study, we explored and compared the in vitro and in vivo activities of recombinant anthrax protective antigen (rPA) and receptor binding domain of protective antigen (PA4). As a result, the fully soluble rPA and PA4 proteins were successfully expressed in Escherichia coli by co-expression with thioredoxin (Trx), and the rPA was active in forming cytotoxic lethal toxins, indicating that the rPA protein retains a functionally biological activity. Furthermore, immunization with rPA protein induced stronger PA-specific immune responses in mice than PA4 protein. The protection elicited by immunization with PA4 suggests the presence of common neutralizing epitopes between rPA and PA4, but the immunization with rPA protein induced stronger neutralizing antibodies and protective levels against challenge with the B. anthracis strain A16R than the PA4 protein. The sera neutralizing antibodies titers correlated well with anti-PA group ELISA antibodies titers and the in vivo protective potency. Based on the results of cell cytotoxicity assays and the observed immune responses and protective potency, we concluded that the soluble rPA protein retains the in vitro and in vivo functionally biological activity and can be developed into a highly effective human subunit vaccine candidate against anthrax.

The Use of Serological Tests (I.F.A.T. and iELISA) for Evaluation of Specific Antibodies Level in Vaccinated and Nonvaccinated Animals Against Anthrax

Various diseases, especially infectious ones, have caused untold harm to humanity in centuries. Among these, special attention should be paid to infectious diseases, especially anthrax, which reduces a lot the number of heads and livestock products. One of the most important tools to combat this infection are vaccinations. Today there are different kinds of vaccines against anthrax. The aim of this study was the use of IFA tests and iELISA to determine the efficacy of vaccines against anthrax. For the implementation of the IFA test bacterial emulsions prepared with bacterial strains of B. anthracis Stern 34F2 and Pasteur were used as antigens. As for determining the level of antibodies in the serum by iELISA was used Anthrax Protective antigen IgG ELISA kit. Sera of sheep and goat vaccinated with capsular and noncapsular vaccines were analyzed with both methods 21 days, 9 months and 12 months after vaccination. The results obtained show that sheep respond better to the vaccine than goat. The index of IgG anti-PA antibodies between vaccines, determined by ELISA, varies from 3.3 to 1.6 and 3.85 to 1.94, respectively, in the sera of animals vaccinated with noncapsular and capsular vaccines 21 days and 12 months after vaccination. For evaluation of vaccine immunogenicity, techniques of IgG anti-PA ELISA and indirect immunofluorescence (IFAT) can be used as alternative methods of control by the State Laboratory of Control of Veterinary Bioproducts. DOI: 10.5901/ajis.2013.v2n9p642

Increased long-term immunity to Bacillus anthracis protective antigen in mice immunized with a CIA06B-adjuvanted anthrax vaccine

Anthrax is an acute infectious disease caused by Bacillus anthracis. We previously reported that the adjuvant CIA06B, which consists of TLR4 agonist CIA05 and aluminum hydroxide (alum), enhanced the immune response to anthrax protective antigen (PA) in mice. This study was carried out to determine whether CIA06B can enhance long-term immune responses to PA in mice. BALB/c mice were immunized intramuscularly three times at 2-week intervals with recombinant PA alone or PA combined with alum or CIA06B. At 8 and 24 weeks post-immunization, the immunological responses including serum anti-PA IgG antibody titer, toxin-neutralizing antibody titer, splenic cytokine secretion and the frequency of PA-specific memory B cells were assessed. Compared with mice injected with PA alone or PA plus alum, mice injected with PA plus CIA06B had higher titers of serum anti-PA IgG antibodies, and higher frequencies of PA-specific memory B cells and interferon-γ secreting cells. Furthermore, anti-PA antibodies induced by CIA06B were more effective in neutralizing anthrax toxin. These results demonstrated that CIA06B is capable of providing long-term immunity when used as an adjuvant in a PA-based anthrax vaccine.

A Novel Mechanism for Antibody-based Anthrax Toxin Neutralization: INHIBITION OF PREPORE-TO-PORE CONVERSION

Protective antigen (PA), a key component of anthrax toxin, mediates the entry of lethal factor (LF) or edema factor (EF) through a membranal pore into target cells. We have previously reported the isolation and chimerization of cAb29, an anti-PA monoclonal antibody that effectively neutralizes anthrax toxin in an unknown mechanism. The aim of this study was to elucidate the neutralizing mechanism of this antibody in vitro and to test its ability to confer post-exposure protection against anthrax in vivo. By systematic evaluation of the steps taking place during the PA-based intoxication process, we found that cAb29 did not interfere with the initial steps of intoxication, namely its ability to bind to the anthrax receptor, the consecutive proteolytic cleavage to PA(63), oligomerization, prepore formation, or LF binding. However, the binding of cAb29 to the prepore prevented its pH-triggered transition to the transmembranal pore, thus preventing the last step of intoxication, i.e. the translocation of LF/EF into the cell. Epitope mapping, using a phage display peptide library, revealed that cAb29 binds the 2α(1) loop in domain 2 of PA, a loop that undergoes major conformational changes during pore formation. In vivo, we found that 100% of anthrax-infected rabbits survived when treated with cAb29 12 h after exposure. In conclusion, these experiments demonstrate that cAb29 exerts its potent neutralizing activity in a unique manner by blocking the prepore-to-pore conversion process.

Characterization of a Therapeutic Model of Inhalational Anthrax Using an Increase in Body Temperature in New Zealand White Rabbits as a Trigger for Treatment

The development of an appropriate animal therapeutic model is essential to assess the potential efficacy of therapeutics for use in the event of a Bacillus anthracis exposure. We conducted a natural history study that showed New Zealand White rabbits exhibited a significant increase in body temperature (SIBT), changes in hematologic parameters, and increases in C-reactive protein and succumbed to disease with an average time to death of approximately 73 h following aerosol challenge with B. anthracis Ames spores. The SIBT was used as a trigger to treat with a fully human monoclonal antibody directed at protective antigen (PA). Ninety percent (9/10) of the treated rabbits survived the lethal inhalational challenge of B. anthracis. Further characterization investigated the protective window of opportunity for anti-PA antibody administration up to 12 h post-onset of SIBT. Eighty-three percent (5/6) of the rabbits treated at SIBT and 100% (6/6) of those treated at 6 h after SIBT survived challenge. Only 67% (4/6) of the rabbits treated at 12 h after SIBT survived. The increase in body temperature corresponded with both bacteremia and antigenemia (PA in the blood), indicating that SIBT is a suitable trigger to initiate treatment in a therapeutic model of inhalational anthrax.

Structural and Immunological Analysis of Anthrax Recombinant Protective Antigen Adsorbed to Aluminum Hydroxide Adjuvant

New anthrax vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on aluminum adjuvant. Intrinsic fluorescence emission spectra of tryptophan residues indicated that some tertiary structure alterations of rPA occurred during storage on Alhydrogel. Using anti-PA monoclonal antibodies to probe specific regions of the adsorbed rPA molecule, we found that two monoclonal antibodies that recognize epitopes located in domain 1 of PA exhibited greater reactivity to the stored formulations than to freshly prepared formulations. Immunogenicity of rPA-Alhydrogel formulations in mice was assessed by measuring the induction of toxin-neutralizing antibodies, as well as antibodies reactive to 12-mer peptides spanning the length of PA. Mice immunized with freshly prepared formulations developed significantly higher toxin-neutralizing antibody titers than mice immunized with the stored preparations. In contrast, sera from mice immunized with stored preparations exhibited increased reactivity to nine 12-mer peptides corresponding to sequences located throughout the rPA molecule. These results demonstrate that storage of rPA-Alhydrogel formulations can lead to structural alteration of the protein and loss of the ability to elicit toxin-neutralizing antibodies.

Fluorescence spectroscopy measures yeast PAH1-encoded phosphatidate phosphatase interaction with liposome membranes

Phosphatidate (PA) phosphatase, the enzyme that catalyzes the penultimate step in triacylglycerol synthesis, is a cytosolic enzyme that must associate with the membrane where its substrate PA resides. Fluorescence spectroscopy was used to measure the interaction of yeast PAH1-encoded PA phosphatase with model liposome membranes. PA phosphatase contains five tryptophan residues and exhibited inherit fluorescence that increased upon interaction with phosphatidylcholine liposomes. The interaction was enhanced by inclusion of other phospholipids and especially the substrate PA. Interaction was dependent on both the concentration of phosphatidylcholine-PA liposomes as well as the surface concentration of PA in liposomes. Mg(2+) ions, which were required for catalysis, did not affect PA phosphatase interaction with phosphatidylcholine-PA liposomes. PA phosphatase was a substrate for protein kinase A, protein kinase C, and casein kinase II, and these phosphorylations decreased PA phosphatase interaction with phosphatidylcholine-PA liposome membranes.

The early humoral immune response toBacillus anthracistoxins in patients infected with cutaneous anthrax

Bacillus anthracis, the causative agent of anthrax, produces a tripartite toxin composed of two enzymatically active subunits, lethal factor (LF) and edema factor (EF), which, when associated with a cell-binding component, protective antigen (PA), form lethal toxin and edema toxin, respectively. In this preliminary study, we characterized the toxin-specific antibody responses observed in 17 individuals infected with cutaneous anthrax. The majority of the toxin-specific antibody responses observed following infection were directed against LF, with immunoglobulin G (IgG) detected as early as 4 days after the onset of symptoms in contrast to the later and lower EF- and PA-specific IgG responses. Unlike the case with infection, the predominant toxin-specific antibody response of those immunized with the US anthrax vaccine absorbed and UK anthrax vaccine precipitated licensed anthrax vaccines was directed against PA. We observed that the LF-specific human antibodies were, like anti-PA antibodies, able to neutralize toxin activity, suggesting the possibility that they may contribute to protection. We conclude that an antibody response to LF might be a more sensitive diagnostic marker of anthrax than to PA. The ability of human LF-specific antibodies to neutralize toxin activity supports the possible inclusion of LF in future anthrax vaccines.

Negatively Charged Liposomes Show Potent Adjuvant Activity When Simply Admixed with Protein Antigens

Liposomes have been investigated extensively as a vaccine delivery system. Herein the adjuvant activities of liposomes with different net surface charges (neutral, positive, or negative) were evaluated when admixed with protein antigens, ovalbumin (OVA, pI = 4.7), Bacillus anthracis protective antigen protein (PA, pI = 5.6), or cationized OVA (cOVA). Mice immunized subcutaneously with OVA admixed with different liposomes generated different antibody responses. Interestingly, OVA admixed with net negatively charged liposomes prepared with DOPA was as immunogenic as OVA admixed with positively charged liposomes prepared with DOTAP. Immunization of mice with the anthrax PA protein admixed with the net negatively charged DOPA liposomes also induced a strong and functional anti-PA antibody response. When the cationized OVA was used as a model antigen, liposomes with net neutral, negative, or positive charges showed comparable adjuvant activities. Immunization of mice with the OVA admixed with DOPA liposomes also induced OVA-specific CD8(+) cytotoxic T lymphocyte responses and significantly delayed the growth of OVA-expressing B16-OVA tumors in mice. However, not all net negatively charged liposomes showed a strong adjuvant activity. The adjuvant activity of the negatively charged liposomes may be related to the liposome's ability (i) to upregulate the expression of molecules related to the activation and maturation of antigen-presenting cells and (ii) to slightly facilitate the uptake of the antigens by antigen-presenting cells. Simply admixing certain negatively charged liposomes with certain protein antigens of interest may represent a novel platform for vaccine development.

A combination of the TLR4 agonist CIA05 and alum promotes the immune responses to Bacillus anthracis protective antigen in mice

Anthrax is an infectious disease caused by Bacillus anthracis. The currently licensed human anthrax vaccines contain protective antigen (PA) as a major protective component and alum as an adjuvant. In this study, we investigated whether CIA05, a TLR4 agonist, is able to promote the immune response to an anthrax vaccine adjuvanted with alum. BALB/c mice were immunized intraperitoneally three times at 2-week intervals with a recombinant B. anthracis PA alone or in combination with CIA05 in the absence or presence of alum, and immune responses were determined 2 or 3weeks after the third immunization. The results showed that the combination of CIA05 and alum significantly increased both serum anti-PA IgG antibody and toxin-neutralizing antibody titers, and the adjuvant effects were greater when lower antigen doses were used for immunization. Both CIA05 and alum stimulated PA-specific splenocyte secretion of interleukin (IL)-4, IL-5, and IL-6. A combination of the two yielded synergistic effects on IL-4 secretion, but CIA05 tended to repress IL-5 and IL-6 secretions induced by alum. Co-administration of CIA05 and alum also increased GL7 expression in B220+CD24+ splenic cells, indicating the ability to activate B cells. These data suggest that CIA05, combined with alum, could be used to achieve higher immune responses to PA, leading to the development of an effective anthrax vaccine.

Anthrax vaccination induced anti-lethal factor IgG: Fine specificity and neutralizing capacity

The efficacy biomarker of the currently licensed anthrax vaccine (AVA) is based on quantity and neutralizing capacity of anti-protective antigen (anti-PA) antibodies. However, animal studies have demonstrated that antibodies to lethal factor (LF) can provide protection against in vivo bacterial spore challenges. Improved understanding of the fine specificities of humoral immune responses that provide optimum neutralization capacity may enhance the efficacy of future passive immune globulin preparations to treat and prevent inhalation anthrax morbidity and mortality. This study (n=1000) was designed to identify AVA vaccinated individuals who generate neutralizing antibodies and to determine what specificities correlate with protection. The number of vaccine doses, years post vaccination, and PA titer were associated with in vitro neutralization, reinforcing previous reports. In addition, African American individuals had lower serologic neutralizing activity than European Americans, suggesting a genetic role in the generation of these neutralizing antibodies. Of the vaccinated individuals, only 69 (6.9%) had moderate levels of anti-LF IgG compared to 244 (24.4%) with low and 687 (68.7%) with extremely low levels of IgG antibodies to LF. Using overlapping decapeptide analysis, we identified six common LF antigenic regions targeted by those individuals with moderate levels of antibodies to LF and high in vitro toxin neutralizing activity. Affinity purified antibodies directed against antigenic epitopes within the PA binding and ADP-ribotransferase-like domains of LF were able to protect mice against lethal toxin challenge. Findings from these studies have important implications for vaccine design and immunotherapeutic development.

Antibiotics Cure Anthrax in Animal Models

Respiratory anthrax, in the absence of early antibiotic treatment, is a fatal disease. This study aimed to test the efficiency of antibiotic therapy in curing infected animals and those sick with anthrax. Postexposure prophylaxis (24 h postinfection [p.i.]) of guinea pigs infected intranasally with Bacillus anthracis Vollum spores with doxycycline, ofloxacin, imipenem, and gentamicin conferred protection. However, upon termination of treatment, the animals died from respiratory anthrax. Combined treatment with antibiotics and active vaccination with a protective antigen-based vaccine leads to full protection even after cessation of treatment. Delaying the initiation of antibiotic administration to over 24 h p.i. resulted in treatment of animals with anthrax exhibiting various degrees of bacteremia and toxemia. Treatment with doxycycline or ciprofloxacin cured sick guinea pigs and rabbits exhibiting bacteremia levels up to 10(5) CFU/ml. Addition of anti-protective antigen (PA) antibodies augmented the efficiency of protection, allowing the cure of guinea pigs and rabbits with 10- to 20-fold-higher bacteremia levels, up to 7 × 10(5) CFU/ml and 2 × 10(6) CFU/ml, respectively. Treatment with ciprofloxacin and a monoclonal anti-PA antibody rescued rabbits with bacteremia levels up to 4 × 10(6) CFU/ml. During antibiotic administration, all surviving animals developed a protective immune response against development of a fatal disease and subcutaneous challenge with Vollum spores. In conclusion, these results demonstrate that antibiotic treatment can prevent the development of fatal disease in respiratory-anthrax-infected animals and can cure animals after disease establishment. A therapeutic time window of 40 h to 48 h from infection to initiation of efficient antibiotic-mediated cure was observed.

Development and validation of serological methods for human vaccine potency testing: Case study of an anthrax vaccine

Only one anthrax vaccine (Anthrax Vaccine Adsorbed, AVA) is licensed in the US to date, although new vaccines are under development. Logistic difficulties with the potency testing of AVA, plus humane considerations, have prompted the development of an alternative test method that can be used as a potency test not only for AVA, but also for newer vaccines. A potency test is not limited to measuring the concentration and quality of the antigen in the final formulation at the time of vaccine release, but should also detect changes in these characteristics during the dating period of the product, to ensure that the vaccine has retained its potency. We have developed a mouse immunogenicity test with potential for use as a potency test for anthrax vaccines. This model is based on the measurement of antibodies induced by a fixed dose of antigen. The test consists of two stages: a) the induction of antibodies in mice with one pre-selected test dose of vaccine; and b) the measurement of the response. We have established the test dose for AVA and experimental vaccines based on the use of anthrax Protective Antigen (PA). Two types of assays, an ELISA and a toxin neutralization assay (TNA) have been employed to measure antibodies to PA. TNA may be more useful in predicting vaccine efficacy, since it measures the neutralizing activity of sera against the cytotoxic effect of the toxin formed by PA when associated with Lethal Factor. However, anti-PA ELISA is less demanding from a technical point of view. Therefore, if ELISA were capable of detecting accurately changes in antigen quantity and quality, then it could be selected as the antibody-measuring test. Studies in our laboratory suggest that even though ELISA and TNA results are correlated, they may not be strictly interchangeable for quantification of anti-PA antibodies after a single immunization of mice with a test dose of anthrax vaccine. We have also found that TNA is better suited than ELISA to detect changes in immunogenicity caused by vaccine exposure to high temperature for very short periods (two minutes). These results open the possibility that an immunogenicity test in which TNA is used to quantify the anti-PA antibody responses can be used to measure vaccine potency of anthrax vaccines at the end of the manufacturing process and periodically after the finished product has been placed in storage, instead of an active protection test that requires lethal challenge.

Progress and novel strategies in vaccine development and treatment of anthrax

The lethal anthrax disease is caused by spores of the gram-positive Bacillus anthracis, a member of the cereus group of bacilli. Although the disease is very rare in the Western world, development of anthrax countermeasures gains increasing attention due to the potential use of B. anthracis spores as a bio-terror weapon. Protective antigen (PA), the non-toxic subunit of the bacterial secreted exotoxin, fulfills the role of recognizing a specific receptor and mediating the entry of the toxin into the host target cells. PA elicits a protective immune response and represents the basis for all current anthrax vaccines. Anti-PA neutralizing antibodies are useful correlates for protection and for vaccine efficacy evaluation. Post exposure anti-toxemic and anti-bacteremic prophylactic treatment of anthrax requires prolonged antibiotic administration. Shorter efficient postexposure treatments may require active or passive immunization, in addition to antibiotics. Although anthrax is acknowledged as a toxinogenic disease, additional factors, other than the bacterial toxin, may be involved in the virulence of B. anthracis and may be needed for the long-lasting protection conferred by PA immunization. The search for such novel factors is the focus of several high throughput genomic and proteomic studies that are already leading to identification of novel targets for therapeutics, for vaccine candidates, as well as biomarkers for detection and diagnosis.

Recombinant Anthrax Toxin Receptor-Fc Fusion Proteins Produced in Plants Protect Rabbits against Inhalational Anthrax

Inhalational anthrax, a zoonotic disease caused by the inhalation of Bacillus anthracis spores, has a ∼50% fatality rate even when treated with antibiotics. Pathogenesis is dependent on the activity of two toxic noncovalent complexes: edema toxin (EdTx) and lethal toxin (LeTx). Protective antigen (PA), an essential component of both complexes, binds with high affinity to the major receptor mediating the lethality of anthrax toxin in vivo, capillary morphogenesis protein 2 (CMG2). Certain antibodies against PA have been shown to protect against anthrax in vivo. As an alternative to anti-PA antibodies, we produced a fusion of the extracellular domain of human CMG2 and human IgG Fc, using both transient and stable tobacco plant expression systems. Optimized expression led to the CMG2-Fc fusion protein being produced at high levels: 730 mg/kg fresh leaf weight in Nicotiana benthamiana and 65 mg/kg in N. tabacum. CMG2-Fc, purified from tobacco plants, fully protected rabbits against a lethal challenge with B. anthracis spores at a dose of 2 mg/kg body weight administered at the time of challenge. Treatment with CMG2-Fc did not interfere with the development of the animals' own immunity to anthrax, as treated animals that survived an initial challenge also survived a rechallenge 30 days later. The glycosylation of the Fc (or lack thereof) had no significant effect on the protective potency of CMG2-Fc in rabbits or on its serum half-life, which was about 5 days. Significantly, CMG2-Fc effectively neutralized, in vitro, LeTx-containing mutant forms of PA that were not neutralized by anti-PA monoclonal antibodies.

Vaccination of Rhesus Macaques with the Anthrax Vaccine Adsorbed Vaccine Produces a Serum Antibody Response That Effectively Neutralizes Receptor-Bound Protective AntigenIn Vitro

Anthrax toxin (ATx) is composed of the binary exotoxins lethal toxin (LTx) and edema toxin (ETx). They have separate effector proteins (edema factor and lethal factor) but have the same binding protein, protective antigen (PA). PA is the primary immunogen in the current licensed vaccine anthrax vaccine adsorbed (AVA [BioThrax]). AVA confers protective immunity by stimulating production of ATx-neutralizing antibodies, which could block the intoxication process at several steps (binding of PA to the target cell surface, furin cleavage, toxin complex formation, and binding/translocation of ATx into the cell). To evaluate ATx neutralization by anti-AVA antibodies, we developed two low-temperature LTx neutralization activity (TNA) assays that distinguish antibody blocking before and after binding of PA to target cells (noncomplexed [NC] and receptor-bound [RB] TNA assays). These assays were used to investigate anti-PA antibody responses in AVA-vaccinated rhesus macaques (Macaca mulatta) that survived an aerosol challenge with Bacillus anthracis Ames spores. Results showed that macaque anti-AVA sera neutralized LTx in vitro, even when PA was prebound to cells. Neutralization titers in surviving versus nonsurviving animals and between prechallenge and postchallenge activities were highly correlated. These data demonstrate that AVA stimulates a myriad of antibodies that recognize multiple neutralizing epitopes and confirm that change, loss, or occlusion of epitopes after PA is processed from PA83 to PA63 at the cell surface does not significantly affect in vitro neutralizing efficacy. Furthermore, these data support the idea that the full-length PA83 monomer is an appropriate immunogen for inclusion in next-generation anthrax vaccines.

Anthrax toxin-neutralizing antibody reconfigures the protective antigen heptamer into a supercomplex

The tripartite protein exotoxin secreted by Bacillus anthracis, a major contributor to its virulence and anthrax pathogenesis, consists of binary complexes of the protective antigen (PA) heptamer (PA63h), produced by proteolytic cleavage of PA, together with either lethal factor or edema factor. The mouse monoclonal anti-PA antibody 1G3 was previously shown to be a potent antidote that shares F(C) domain dependency with the human monoclonal antibody MDX-1303 currently under clinical development. Here we demonstrate that 1G3 instigates severe perturbation of the PA63h structure and creates a PA supercomplex as visualized by electron microscopy. This phenotype, produced by the unconventional mode of antibody action, highlights the feasibility for optimization of vaccines based on analogous structural modification of PA63h as an additional strategy for future remedies against anthrax.

Evaluation of the immune response induced by a nasal anthrax vaccine based on the protective antigen protein in anaesthetized and non-anaesthetized mice

To better protect against inhalational anthrax infection, a nasal anthrax vaccine based on the protective antigen (PA) protein of Bacillus anthracis could be an attractive alternative to the current Anthrax-Vaccine-Adsorbed (AVA), which was licensed for cutaneous anthrax prevention. Previously, we have demonstrated that an anti-PA immune response comparable with that in mice subcutaneously immunized with PA protein adjuvanted with aluminium hydroxide was induced in both the systemic compartment and the mucosal secretions of the nose and lung of anaesthetized mice when they were nasally immunized with PA protein incorporated into previously reported LPD (Liposome-Protamine-DNA) particles. In this study, we evaluated the anti-PA immune response induced by the nasal PA/LPD particles in non-anaesthetized mice and compared it with that in anaesthetized mice. Our data showed that the anti-PA antibody response and the anthrax lethal toxin-neutralization activity induced by the nasal PA/LPD in non-anaesthetized mice was relatively weaker than that in anaesthetized mice. However, the splenocytes isolated from the nasally immunized mice, anaesthetized and non-anaesthetized, proliferated comparably after in-vitro re-stimulation. By evaluating the uptake of fluorescence-labelled LPD particles by phagocytes in the nasal and broncho-alveolar lavages of mice after the nasal administration, we concluded that the relatively weaker anti-PA immune response in the non-anaesthetized mice might be partially attributed to the reduced retention of the PA/LPD particles in the nasal cavity of the non-anaesthetized mice. Data collected in this study are expected to be useful for future anthrax nasal vaccine studies when mice are used as a model.

Intérêt des anticorps recombinants dans la protection médicale contre les agents du bioterrorisme : l'exemple de la maladie du charbon

Recombinant antibodies are a highly successful class of therapeutic molecules, they are well adapted for use against bio-weapons (BW) as they act immediately, are often synergistic with other therapeutic molecules, have a long half-life and are well tolerated. Anthrax is regarded at high risk of being used as BW, and its pathogenic properties depend on toxins, which might be neutralized by antibodies. These toxins are made of three different types of sub-units (PA, LF, EF). Several anti-PA have been developed, including an original approach by our team. We have developed an anti-LF, as recommended by experts. Our anti-PA antibody, and to a lesser extend our anti-LF antibody, will be presented here.